Our lab has been studying the role if specific phosphorylation site, serine 1179 in regulating eNOS function and that the protein kinase Akt1 is necessary for post-natal angiogenesis in response to hindlimb ischemia or to exogenous administration of vascular endothelial growth factor (VEGF). In this competitive renewal, we will examine the role of eNOS phosphorylation on S1176 in macro- and micro-vascular functions of endothelial derived NO, namely atheroprotection and angiogenesis, respectively. Thus, we hypothesize that the phosphorylation of eNOS governs the production of NO to regulate macrovascular (atheroprotection) and microvascular (angiogenesis) functions in the cardiovascular system. To test these hypotheses, we propose the following Specific Aims: 1.Determine the mechanisms of why the loss of Akt1 promotes coronary atherogenesis. We will explore potential mechanisms in vitro in isolated macrophages and vascular cells, in vivo by bone marrow transplantation experiments and by breeding eNOS phosphomutant mice to the ApoE/Akt1 null background;2. Dissect the role of endothelial specific Akt1 regulating macrovascular (atherogenesis) and microvascular (angiogenesis) function by conditional deletion of Akt1 in endothelial cells;3 Decipher the importance of eNOS phosphorylation on serine 1176 using knockin mice expressing either constitutively active (S1176D eNOS) or less active eNOS (S1176A eNOS). The importance of eNOS as a key Akt substrate will be tested in vitro and in several postnatal models of angiogenesis in mice replete or deficient in Akt1. Collectively, this work will facilitate our understanding of the importance of eNOS phosphorylation in atherogenesis and angiogenesis and provide insights into the molecular machinery required for eNOS regulation using a multi-disciplinary approach. PUBLIC HEALTH RELEVANCE This research is relevant to public health since endothelial dysfunction is a common manifestation of most cardiovascular diseases. Our research has discovered the major mechanisms of how the endothelium control blood flow and atherogenesis. Research supported by this grant may help identify new drugs that reduce heart disease and improve the quality of life of people suffering with cardiovascular disease.